SUMMARY Amyotrophic lateral sclerosis (ALS) is a mostly sporadic and invariably fatal paralytic disorder. ALS pathogenic mechanisms are elusive and its etiology is poorly understood, even if environmental exposures, including toxic metals, are thought to play a key role. Progresses in both therapy and prevention for ALS have been held back by the lack of clear biomarkers, indicative of patients' disease progression and central nervous system (CNS) exposure to environmental toxicants. Here, we propose to test whether the cargoes of blood extra-cellular vesicles (EVs), i.e., tiny membrane-bound capsules that shuttle biomolecules out of the CNS, can serve as novel biomarkers of CNS metal exposure and disease progression in ALS. EVs carry nucleic acids (including microRNAs [miRNAs]), proteins, and other elements, while circulating throughout the body for inter- organ communication. EVs also operate as trash bags allowing cells to eliminate excesses of unwanted cellular materials, including toxic metals and proteins. CNS-EVs can be isolated from blood because they express on their surface the neuron-specific protein L1CAM or the astrocyte-specific protein GLAST. Blood CNS-EVs could open a direct window of observation into the ALS brain from the periphery and provide different types of biomarkers. First, CNS-EVs may reflect disease progression by carrying pathogenic proteins progressively accumulating in neurons and astrocytes in ALS, such as the TAR DNA-binding protein 43 (TDP-43), or by showing progressive changes in miRNA profiles. Second, CNS-EV metal levels could be a direct surrogate of CNS metal load, and as suggested by metals' effects on miRNA profiles, they could provide a molecular fingerprint of patient metal exposure, when direct measurement is not possible. To test these hypotheses, we will leverage unique biospecimen collections from three well-phenotyped cohorts of ALS patients and a set of 140 control samples: (1) the COSMOS multi-center cohort (n=269 patients; 2-6 serial visits; blood); (2) the NEALS longitudinal study for ALS biomarkers (LABB) (N=150) with 2-3 visits; and (3) the Veterans Affairs Biorepository (VAB) (n=147; paired blood and postmortem CNS tissues). Clinical stage was assessed at each blood draw through the ALS Functional Rating Scale-Revised. Our specific aims are to: Aim-1: Test whether TDP-43 level in blood L1CAM/GLAST-EVs is a biomarker of ALS progression by ELISA quantification of TDP-43 in the serial COSMOS and LABB blood draws, and testing its association with ALS progression. Aim- 2: Assess metal content in blood CNS-EV as a biomarker of patient's exposure by measuring the levels of 7 metals linked to ALS in paired CNS-EVs and CNS tissues (VAB), and testing their association with ALS progression (LABB). Aim-3: Test whether miRNA profiles in blood CNS-EVs are biomarkers of both ALS progression and metal exposure by miRNA-sequencing and assessing whether they mediate TDP-43's and metals' effect on ALS progression. The novel and minimally invasive EV biomarkers tested here could greatly improve the evaluation of ALS treatment efficacy, and revolutionize metal exposure assessment.